The large fragment of DNA polymerase I, isolated from Bacillus stearothermophilus, was used for dideoxy sequencing. This heat-stable enzyme permits performing sequencing reactions at high temperature to melt secondary structure and results in uniform band intensities and low background on the autoradiogram. The enzyme can be used in the standard Sanger one-step protocol or in a two-step protocol which separates the labeling reaction from the elongation-termination reaction. The enzyme can be used in double-stranded sequencing. 35S-labeled nucleotides may be used instead of 32P-labeled nucleotides. Both 7-deaza-dGTP and dITP can be used during the reaction in order to minimize band compression on the gel. Results presented here indicate that this enzyme should be a useful tool for sequence determination.